Fig 2: Over-expression of SAFB1 leads to formation of enlarged, thicker, MHC-positive myotubes. (A) C2C12 cells infected with the shRNA control vector, an shRNA against Safb1 or a vector encoding SAFB1 were immunostained for MHC after 48 and 72 h of differentiation. Nuclei were counterstained with DAPI. (B) Western blot of protein extracts obtained from duplicate plates of cells in (A) showing Safb1 and MHC levels. PI3K levels were monitored as a loading control. (C) Immunostaining indicates that exogenous expression of SAFB1 in Safb1 knockdown cells rescues MHC expression and results in myotube formation similar to that observed in sh-control cells over-expressing SAFB1. Nuclei were counterstained with DAPI. (D) Rescue of Safb1 expression after knockdown of endogenous Safb1 (shSafb1 + SAFB1), reestablishes MHC expression at 48 h of differentiation. HA signal indicates the presence of exogenous SAFB1, which is HA-tagged. PI3K levels were monitored as a loading control.

Fig 3: shRNA-mediated knockdown of Safb1 does not alter MyoD recruitment to myogenic sequences but increases Polycomb repressive marks. Proliferating (GM) or 72 h differentiated (DM) C2C12 cells expressing control shRNA or shRNA against Safb1 were fixed and processed for ChIP assays using (A) antibodies against MyoD and Safb1 (top panel) or (B) antibodies against Ezh2 and H3K27me3 at the indicated gene regulatory sequences. H3K27me3 enrichment is presented on the right-hand y-axis in the bottom panel. Data represent the mean of three independent experiments ± SEM. t test analyses indicate that when comparing binding in sh-Safb1-treated differentiated cells with sh-Control differentiated cells, the binding of Safb1 was significantly reduced (P < 0.01), the binding of MyoD was not significantly reduced, except for the binding to the MyhIIb promoter (P = 0.045), and the binding of Ezh2 and H3K27me3 were significantly reduced (P < 0.01 and P < 0.005, respectively).

Fig 4: SAFB and SAFB2 safeguard host gene transcription by rectifying the transcription blocking effects of MILs.a RT-qPCR results showing the expression changes of L1HS after knocking down SAFB or SAFB2 in K562 cells by siRNA. The mRNA levels of SAFB and SAFB2 are also shown. siSAFB or siSAFB2 indicates single knockdown; siSAFB&B2 indicates dual depletion. b A heatmap showing the RNA abundances of different L1 sub-families after depleting SAFB (siSAFB) or co-depleting SAFB and SAFB2 (siSAFB&B2) by siRNAs. Data are from RNA-Seq and indicate Log2 fold changes of expression in the knockdown group as compared to siCTL group. c Box plot showing the TBIs (see Fig. 5c) of Control L1, Super-MILs or typical MILs on their hosting genes. Data are based on TT-Seq in K562 cells with the same groups of knockdown as in panel b. P-values were calculated with a paired Student’s t-test. d Genome browser snapshot showing TT-Seq signals over the PSMA1 (upper) or ZRANB3 gene loci (lower) in control or specific knockdown cells as indicated. The MINT-Seq track on top of each plot indicates m6A signals for strong Super-MILs. The dash lines denote the 3’ end of the Super-MIL regions. Yellow highlights point to regions with strong transcriptional change. TBI of Super-MIL in each TT-Seq is labeled. RT-qPCR result showing mRNA reduction of ZRANB3 (e) and PSMA1(f) after depletion of SAFB (siSAFB) or co-depletion of SAFB and SAFB2 (siSAFB&B2) in wild type (WT) K562 cells or corresponding Super-MIL knockout cells. g Heatmaps generated by analyzing RNA-seq of siCTL, siSAFB and siSAFB&B2 K562 cells, showing the fold changes of MIL-hosting genes that are DDR genes. Scale bars are shown on the right (i.e., downregulated genes are in blue colors). h Similar to panel g, but these plots show fold changes of MIL-hosting genes that are putative L1 suppressors (by Liu et al.,63 left panel; and by Mita et al.,11 right panel). i A regression plot showing the relationship between gene length and their expression changes after SAFB or SAFB&B2 depletion in K562 cells. MIL-hosting genes were divided into five equal-numbered groups by length (x-axis), and the fold changes of each group are shown (the dots indicate mean fold changes for genes of each group, and vertical lines denote 95% confidence interval of mean fold changes). The lines show linear regression and the shaded areas of matched colors denote a 95% confidence interval for that regression. There are 422 long genes (> 200 kb) harboring MILs. Data of siSAFB or siSAFB&B2 in this figure were based on polyA selected RNA-Seq. For all qPCRs, data show means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t-test.

Fig 5: Opposite roles of m6A modification and SAFB on L1 expression and retrotransposition.a A heatmap showing the RNA expression changes of L1 sub-families after knocking down target proteins (based on re-analysis of ENCODE K562 RNA-Seq data). Fold changes were based on comparison to respective sgRNA or shRNA controls. b, c Heatmaps showing the Log2 fold change of L1 RNA abundances (b, measured by ribo-depleted total RNA-Seq) and m6A ratio (c, measured by FPKM of MeRIP-Seq / FPKM of RNA-Seq) of different L1 sub-families after co-depletion of METTL3 and METTL14 by siRNAs (siMETTL3/14). RNA-Seq after transfection by a scramble control siRNA (siCTL) was used as control. d A line plot showing RNA stability of L1HS after flavopiridol treatment for the indicated time. RNA abundance was calculated by RT-qPCR and normalized to 0 h time point in each group. e Normalized RNA expression levels of an active L1HS-Ta (Chr22-q12.1) after depletion of METTL3, YTHDC1 and SAFB by siRNA in MCF7 cells. f Bar plot showing the normalized L1-Neo retrotransposition activity in cells with specific target proteins depleted by siRNAs. Cell colony numbers were counted and compared to the control group. Representative pictures of cell colonies growing in a culture dish are shown on the right. g Sequence comparison between the L1 consensus reporter construct (Con, gray bars) and an L1 RRACH mutant construct (Mut, blue bars). The numbers of RRACH motif in each 500 bp bin throughout the L1HS are shown. h RT-qPCR following m6A RIP (meRIP) showing the relative m6A levels of reporter L1HS RNAs (the Con was set as 1). m6A levels were normalized with synthetic m6A-RNA spike-in. i Similar to panel d, RNA stability of Con L1HS and Mut L1HS RNAs was measured. j Similar to panel f, L1-Neo reporter assay showing the retrotransposition activity of Con L1HS and Mut L1HS. k UV-RIP assay using a native antibody against SAFB showing the normalized binding between SAFB and Con / Mut L1HS RNAs. l Expression changes of Con L1HS or Mut L1HS RNAs after knocking down SAFB. All qPCR data show means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t-test.